Automated ELISA Systems

The ELISA experiment can be broken down into a series of four basic steps dispensing, incubation, washing and reading. The number of individual steps, the order in which they are performed, and the number of repetitions will vary depending on the specific protocol. As such, ELISA is readily amenable to automation. Commercial microplate-based devices have been developed to accomplish the following basic steps (1) automatic plate washers, (2) liquid handlers or microplate dispensers, (3) plate stacks (ambient) or automated incubators, and (4) microplate readers. 

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Miyaguchi, H., Tokeshi, M., and Kikutani, Y., et al. J. Chromatogr. A 1129,105-110 (2006). Ohashi, T., Mawatari, K., Sato, K., et al. Automated micro-ELISA system for allergy checker a prototype and clinical test, in "Proceedings of Micro Total Analysis Systems 2006" (T. Kitamori, H. Fujita, S. Hasebe Eds.), pp. 858-860. Japan Academic Association Inc., Japan (2006). 

Speed of Analysis. The speed with which many immunochemical analyses can be completed illustrates a major advantage of immunochemical procedures. Immunochemical assays are most time and cost effective when the sample load is large. Parker (4) estimated that a single technician could perform 100-5000 radioimmunoassays per day with little or no assay automation in comparison to 20-40 GLC assays (3). Numerous inexpensive systems are available to decrease analysis time. These systems may include solid phase separation techniques, automatic dispensers, test tube racks which will fit directly into a centrifuge and/or scintillation counter, and data handling systems. Alternatively, there are fully automated systems based on RIA or ELISA which require very little operator attention and which handle 25-240 samples/hr. Gochman and Bowie (81) have outlined the basis of operation and summarized the features of automated RIA systems and extensive literature is available from the manufacturers. 

An example of a commercial LoaD platform based on FLISA is the GyroLab workstation (Gyros AB, Uppsala, Sweden) [5]. Lee et al. have reported a portable, fully automated lab-on-a-disc-based ELISA system to test infectious diseases from whole blood [6] which utilizes an optical detection module made up of two sets of matched LEDs and photodiodes to perform absorbance measurements at 430 and 630 nm, respectively (Fig. 2). 

Spectrophotometric plate readers Perkin-Elmer s lambda reader, an automated microprocessor-controlled, microplate reader, offers the flexibility of configuring a reliable, user-friendly, versatile system, capable of accommodating a wide variety of assays requiring calorimetric measurement on microscale (<300pl) samples. These assays include ELISA, protein determination, cytotoxicity, cytoproliferation and antibody sensitivity testing. 

Beckman Robotic Biomek 1000 automated laboratory The Biomek 1000 integrates the work formerly done by four instruments sample preparation system, diluter/dispenser, plate washer and a spectrometer finish. In can handle assays such as radio-immunoassays (RIA), fluorescence immunoassays (FIA), enzyme immunoassays EIA and enzyme-linked immunoassays (ELISA). 

This author and coworkers at Beckman Coulter first described the use of a low form 96-well plastic microplate for automated micro-ELISA immunoassays (Matson et al., 2001). The polypropylene plate was first modified by a radiofrequency plasma amination process (Matson et al., 1995) followed by conversion to an acyl fluoride surface chemistry for rapid covalent attachment of biomolecules. Proteins (1 to 2 mg/mL) were prepared in 50 mM carbonate buffer, pH 9, containing 4% sodium sulfate (to improve spot uniformity) and printed using a conventional arrayer system. Approximately 200-pL droplets of monoclonal antibodies (anti-cytokine) were deposited into the bottom of the microwells using a Cartesian PS7200 system equipped... 

The trend in the detection system employed is more towards automation. A CREAM video image processing system developed by Brogan and co-workers (78) enables one to evaluate and store ELISA data for further statistical manipulation. The CREAM EIA software gives an accurate estimation of sample concentration. It can also be used for quantitation of DOT-blots. 

Cost Effectiveness. As with the other advantages of immunochemical analysis, cost may be quite variable. Reagent costs for several automated systems have been estimated at under 1.25 per sample. The cost is obviously much lower for less sophisticated assay systems, especially if some reagents are prepared in house. A major consideration is the expense of new instrumentation. For dedicated or automated instrumentation for either RIA or ELISA procedures, the cost may be 50-100,000. However, most analytical laboratories already have the basic instrumentation needed for immunoassays. Moderate sensitivity can be obtained through the use of numerous procedures such as radial immunodiffusion and hemagglutination. These procedures require no expensive equipment or reagents and they may be very useful in areas where equipment acquisition or maintenance is a problem. 

Southern [DNA] (32) blots). Recently, a technique has been described for treating nucleic acid target-probe complexes as antigens in an antigen capture system performed in plastic ELISA plates (38). The latter technique is of interest because it allows automated equipment designed for performing standard ELISA to be used for nucleic acid hybridization techniques. 

Fig. 6. The experimental setup for performing automated flow injection-ELISA measurements. FI, excess flow of main buffer F2, main buffer flow F3, washing buffer F4, excess flow of washing buffer F5, substrate solution F6, sample solution F7-F9, waste streams PI and P2, peristaltic pumps VI and V2,3-way valves V3 and V4, injector valves. The computer logs and evaluates the data and controls the flow through the whole system by controlling valves and pumps. (From Nilsson et al. (143).

However, in the long term, ELISA is an ephemeral format. Even when streamlined and automated, it has too many steps. Certainly we should realize that it will be replaced by other systems, the most exciting of which will be biosensors. Also, other formats offer a proprietary edge in the market place which will be very important in the maturation of immunoassay systems in the environmental field. Finally, different formats will lend themselves to different environmental problems. We should continually emphasize that the same reagents can be used in many formats. Possibly in small letters we also should caution that certain antibody characteristics may be more important in one format than another, that some formats are more resistant to matrix effects, and that relative cross reactivities of compounds can change as one changes the subtle principles upon which an immunoassay works. For this reason a clear choice of formats should be made before initiating validation studies. 

A nonisotopic ELISA method in which serum specimens are added to microtiter wells coated with human Tg is also available. In this method, antibody binding is assessed using a peroxidase-conjugated anti-IgG/o-phenylenedi-amine system. An automated two-step fluorescent enzyme immunoassay has been reported. In this assay, Tg is immobilized on magnetic beads, and anti-human IgG mouse monoclonal antibody is labeled with alkafine phosphatase 4-methyiumbelliferyl phosphate is used as the substrate. IRMA and ELISA both have similar detection limits (approximately 3 to 5 U/mL). A considerably more sensitive radioassay has been reported in which diluted serum is incubated with T-labeled Tg to allow formation of antigen-antibody complexes these complexes are then precipitated by adding solid-phase protein A. Its detection limit is reported to be approximately 0.2 U/mL. 

Ruitenberg et al. (1976) and Ruitenberg and Brosi (1978) described an on-line system for macro-ELISA (EIA in disposable polystyrene tubes, 50 X 11 mm) for the daily processing of 4000 samples. Details on automation can be found in the reviews of Ruitenberg and Brosi (1978) and of Carlier et al. (1979). 

As E. coli 0157 H7 is the most implicated STEC serotype in human cases, numerous immuno-based methods for the detection of this specific serotype have been documented. They include conventional Enzyme Linked Immuno Sorbent Assays (ELISA) in microplates, one-step immuno-detection systems, fully automated systems and various non enzymatic immunological based systems like Rapid Plate Latex Agglutination (RPLA), Immuno-Magnetic Separation (IMS) or immuno-chromatography (Table 3). 

A fully automated system derivated from ELISA method has been evaluated by Vemozy-Rozand et al. (1998), tested by the Association of Agricultural Chemists (AOAC) and validated by the Erench national organization for standardization (AENOR). This system, the VIDAS E. coli 0157 (BioMerieux, France) is an Enzyme Linked Fluorescent Assay (ELEA) using two ready-to-use components ... 

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